A schematic of the clotting cascades is shown in FIG. 1(A). In the figure the various clotting factors are indicated by their Roman numeral (i.e., factor VII is indicated by VII). The intrinsic pathway (also referred to as the contact pathway of blood coagulation) is initiated when contact is made between blood and certain artificial surfaces. The extrinsic pathway (also referred to as the tissue factor pathway of blood coagulation) is initiated upon vascular injury which leads to exposure of tissue factor (TF) (also identified as factor III). The dotted arrow represents a point of cross-over between the extrinsic and intrinsic pathways. The two pathways converge at the activation of factor X to Xa. Factor Xa has a role in the further activation of factor VII to VIIa. Active factor Xa hydrolyzes and activates prothrombin to thrombin. Thrombin can then activate factors XI, VIII and V furthering the cascade. Ultimately, the role of thrombin is to convert fibrinogen to fibrin, which forms clots.
Fibrinogen is the most abundant coagulation protein in blood. The formation of a fibrin clot from fibrinogen is the terminal step in the coagulation cascade. Soluble fibrin monomers, which are created when thrombin cleaves fibrinogen, spontaneously polymerize to form a three dimensional network of insoluble fibrin fibrils. Clotting of fibrinogen by thrombin is one of the few steps in the clotting cascade that does not require calcium ions. The resulting fibrin clot structure can be further stabilized via covalent cross-linking of the fibrils through the action of the transglutaminase enzyme, factor XIIIa (FIG. 1 (B)) [26].
Fibrin sealant, also referred to as “fibrin glue” or “fibrin tissue adhesive,” is a surgical hemostatic agent derived from plasma coagulation proteins. Fibrin sealants are widely used to control bleeding in a variety of surgical settings, and their use has increased due to the advent of minimally invasive surgical procedures which necessitate meticulous hemostasis for adequate visualization of the surgical field [27]. Fibrin sealants can be used for hemostasis, wound closure, and tissue sealing and have been advocated as the agents that are closest to approaching the ideal operative sealant. In contrast to synthetic adhesives, fibrin sealants have the advantage of being biocompatible and biodegradable, and they are not associated with inflammation, foreign body reactions, tissue necrosis, or extensive fibrosis. Reabsorption of the fibrin clot is achieved during normal wound healing within days to weeks of application, depending on the type of surgery, the proteolytic activity of the treated site, and the amount of sealant used.
Fibrin sealants are typically derived from plasma proteins and contain two primary components: fibrinogen and thrombin. These two components are stored separately and are mixed during application, whereupon the applied mixture forms a fibrin clot on the wound surface to prevent further hemorrhage. The sealant may be applied with a needle, as a spray, or using other devices. When fibrinogen and thrombin are mixed (during application of fibrin sealant to a wound), the fibrinogen component is converted to fibrin monomers. Polymerization of fibrin monomers results in the formation of a semi-rigid fibrin clot that is capable of interacting covalently and non-covalently with tissue structures. The clot may be further stabilized by cross-linking of the fibrin alpha and gamma chains in a reaction catalyzed by activated factor XIII. This cross-linking stimulates adherence of fibroblasts and promotes their normal growth into the clot. By mimicking the latter stages of the physiologic coagulation system, these processes allow fibrin sealants to arrest blood loss and assist the wound healing process.
Most commercially available fibrin sealants contain purified, virally inactivated human fibrinogen and either human or bovine thrombin, optionally with different quantities of factor XIII and anti-fibrinolytic agents (such as bovine aprotinin). Some of the currently available fibrin sealants are summarized in Table 1. Both Tisseel and Beriplast P are marketed as a two-component kit: component one contains lyophilized pooled human fibrinogen/factor XIII concentrate, which is reconstituted with antifibrinolytic solution (aprotinin); and component two is bovine thrombin reconstituted with 40 mM CaCl2. Tisseel is supplied as a lyophilizate or frozen, whereas Beriplast P is supplied as a lyophilizate. The two-component fibrin sealant is usually applied through a double barreled syringe system, which allows simultaneous application of equal volumes of the fibrinogen and thrombin through a blunt-ended needle or spray tip. Virus inactivation of fibrinogen and thrombin is carried out by a variety of methods, including two-step vapor heat at 60° C. and 80° C., pasteurization (liquid solution, 10 hours at 60° C.), or solvent-detergent treatment, with pasteurization, nanofiltration, or exposure to ultraviolet light.
TABLE 1Composition of fibrin sealantsHumanHumanHumanfactoror bovineBovinefibrinogenXIIIthrombinaprotininSealantForm(mg/mL)(U/mL)(IU/mL)(KIU/mL)Tisseel ®, Tissucol ®Frozen70-11010-505003,000(Duo Baxter-ImmunosolutionAG, Austria)Tisseel ®, Tissucol ®Lyophilizate70-11010-505003,000(Kit Baxter-ImmunoAG, Austria)Tisseel ® (VHLyophilizate75-1155003,000Kit Baxter-ImmunoAG, USA)Beriplast P ® (AventisLyophilizate90605001,000Behring, Germany)(65-115)(40-80)(400-600)Hemaseel ® (APRLyophilizate75-1155003,000Haemacure, Canada)(As Tisseel VH KitBaxter-Immuno)Quixil ®Frozen60-100None1,000 None(Omrix Biopharmaceuticalssolution(tranexamicSA, Israel)acid 92mg/mL)Bolheal ®Lyophilizate 80752501,000(KaketsukenPharmaceutical,Japan)Biocol ® (LFB-Lille,Lyophilizate127115583,000France)VIGuard F.S. ® (Vitex:Lyophilizate50-95 3-5200NoneVI Technologies, USA)
Previous studies showed that polyP shortens the clotting time of human plasma by acting at two steps in the clotting cascade: (a) activating the contact pathway of blood clotting, and (b) accelerating the conversion of factor V to Va [20]. Since polyP did not shorten clotting times when thrombin was added to plasma, it was previously concluded that polyP exerts its procoagulant effects at points in the clotting cascade upstream from thrombin.